The postgenomic era of fluorescence microscopy: molecules, cells and tissues
Oncological research has been deeply involved in this change process, recognising the risks of a sterile approach based solely on in vitro models. The need for therapies targeting specific molecules realises a trait d’union starting from analysis of single molecule biochemical structures and extending to their role in the cellular environment both at intra and intercellular level. The analysed scenario subsequently evolves from clonal tumour populations to the intricate network of interactions established between the tumour and the host first examined in tissue biopsies and then in living animal models.
Modern optical microscopy has always represented an indispensable tool for biomedical research and has developed to adapt to the continuously changing requests dictated by the great heterogeneity of samples targeted by experimental approaches. Besides a someway obvious need to progressively increase resolution, optical microscopy must now extend its non invasive analytical ability from single cells to entire organism minimising the disturbance to life during the observation step.
The revolution of the multiphoton excitation microscope represented a big step toward the observation of complex living systems . Infrared high frequency sources provided an incomparable penetration depth. The ability to easily induce fluorescence from UV excitable molecules has been successfully employed to monitor concentration and functionality of metabolic and structural markers in vivo, such as NADH and collagen respectively. The developed assays supported the pioneer involvement of twophoton microscopes in clinical trials to realise a first approach to “optical biopsies” in the diagnosis of skin neoplasia .
One of the most relevant and successful transformations of the modern microscope is the ability to parallel an instrumental modification with the development of new assays for the determination of functional parameters. Besides the emerging field of nanoscopies, characterised by the birth of 4Pi and STED microscopes allowing the reduction of the gap in spatial resolution between optical and electron microscopy, our insights into the properties of single molecular species gained relevant advantages from the birth of new functional microscopy assays and techniques. This process culminated in the rediscovery of the "F Techniques", namely Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) for the study of molecular dynamics and interactions.
In the following, we will focus our attention on specific applications, providing some examples of how a "standard" confocal microscope can vehicle oncological research across growing levels of complexity. Starting from the properties of single isolated molecules, we will then move to their role in the living-cell biochemical network, scaling up from statically homogeneous samples (in vitro cell cultured populations) to the intrinsic heterogeneity of the histological analysis.
Fig. 1: In vitro fluorescence recovery after photobleaching for characterisation of chemical reaction kinetics (courtesy of A. Musacchio, M. Vink). A: Temporal evolution of the concentrations of the components in a chemical binary system is modelled by differential equations with straightforward analytical solution under proper experimental conditions. One of the two species is immobilised on a surface (coverslip, agarose beads) while the other is covalently tagged with a fluorescent molecule. Formation of the complex is accompanied by accumulation of a fluorescence signal on the coverslip. The temporal behaviour of the fluorescence intensity is ruled by association and dissociation rates depending on the concentration of ligand in solution. B: Recovery of fluorescence after photobleaching of the surface is completely ruled by dissociation rates under the assumptions of no-rebinding and fast diffusion when compared to chemical kinetics. Half-recovery time coincides with the half-life of the studied molecular complex. C: The graph reports the evolution of the intensity of the fluorescence signal. The first phase is characterised by reaching of chemical equilibrium. After that, complexes on the surface were bleached and recovery observed. The different temporal behaviour of the two phases can be consequently appreciated.
Molecules: functional nanoscopies. Characterization of chemical reaction kinetics
The study of complex biochemical networks starts from simplified experimental models as in vitro binary systems constituted by purified biomolecules. Till now, measurements of chemical reaction kinetics parameters were obtained by isothermal calorimetry and/or surface plasmon resonance based instrumentation, with high acquisition and maintenance costs.
Confocal microscopy and, in particular, Fluorescence Recovery After Photobleaching (FRAP) protocols can provide an efficient alternative as recently published in a paper on the characterisation of the molecular mechanisms at the base of the "Spindle Assembly Checkpoint" . In FRAP protocols photobleaching is employed to inactivate fluorescence molecules inside target regions. Molecular mobility leads to recovery of the fluorescent signal by replacement of the bleached fluorescence molecules. The speed of signal recovery represents an index of molecular mobility determined by the diffusion coefficients in the environment where molecules reside (the different cellular compartments) and by the interactions taking place with other molecular species. When a single molecular species is immobilised on a surface and exposed to a second interacting molecule fluorescently tagged in solution, an increase of emitted signal will be detected till reaching of the chemical equilibrium (Figure 1).
The temporal evolution of the intensity will be ruled by the association and dissociation rates proportionally to the concentration of the reactants in the system. However, the recovery of fluorescence in a photobleached region of the above mentioned surface will no longer depend on such parameters. Under proper assumptions, i.e. that the diffusion process took place on a time scale much faster than the chemical reaction, the limiting step in the replacement of a bleached molecule is the disassembly of an existing complex. Half-recovery time consequently coincides with the half-life of the studied molecular complex. Comparison of the graph of the fluorescence temporal evolution revealed the differences of kinetics of the two processes: reaching of chemical equilibrium happened on a faster time scale, while photobleaching did not disturb the gained chemical equilibrium and is consequently ruled out by the half life of the existing complex only.
Cellular networks: highlighting molecular fluxes
The living cell provides a perfect target for validation of FRAP protocols. Coordination of the mechanisms ruling life is accomplished through a perfect degree of spatial and temporal compartmentalisation. Besides FRAP techniques, our ability to follow molecular motion gained significant advantages with the cloning of the photoactivatable/photoconvertible variants of fluorescent proteins. The possibility to selectively switch on fluorescence, creating a highly localised increase in signal-to-noise ratio, enormously facilitated molecular and cellular tracking in living cells and organisms. A strong limitation in photobleaching/photoactivation protocols based on confocal microscopy derives from the inability to control the depth of the targeted volume: a focused Gaussian laser beam will essentially deliver high energy density sufficient to photoactivate/photobleach molecules inside the illumination cone of the objective spanning over several microns.
The use of optical setups with intrinsically limited excitation opened the way to full control of the photoconversion process in three dimensions [4, 5 ]. Total Internal Reflection Fluorescence Microscopy (TIRF) is able to photoactivate molecules on a cell region spanning some hundreds of nanometres over the basal membrane employing the energy delivered by an evanescent electromagnetic field created at the interface between two media with different optical properties, namely the glass coverslip and the cell membrane. Two-photon microscopy is able to move inside the cell, targeting volumes down to a diffraction limited extent. The possibility to confine the activated volume in three dimensions provided new tools for some challenging questions in living cell analysis. Targeted photoactivation of endocytic vesicles without involvement of the upper and lower membrane, only feasible with two-photon induced photoconversion, can be successfully employed to demonstrate the existence of recycling molecular fluxes directed from the cytoplasm to the cell membrane.
Tissues: confocal histopathology in three dimensions
Classical histological analysis has been based for years on the optical microscope, entering only marginally the field of fluorescence microscopy. The evolution of modern confocal microscopy and in particular the birth of spectral detection systems, the high degree of automation and the nonlinear excitation revolution recently stimulated and strengthened interactions between Laser Scanning Confocal Microscopy and histological analysis . Histopathological samples represent a first step towards a rescaling in the complexity of the analysed material required by the postgenomic revolution. This complexity turns the richness of material into a hard environment for light propagation. Light scattremely useful when analysing such complex samples, namely spectral deconvolution. Measurement of the photophysical features of he analysed material can help both in understanding tissue composition and in setting up the best fluorophore combination for maximisation of the analysable parameters in a high content analysis.
The next step consists in obtaining a diffraction limited observation for determination of intracellular parameters over extremely wide fields of view allowing for the recognition of the distinctive tracts of the tissue organisation. The realisation of such a challenging task requires a mosaic approach that can be exploited either by use of automated stages or by intelligent use of the scanning mirrors of the confocal microscope. Such an approach has been adopted to measure the size distribution of ProMyelocytic Leukemia protein nuclear bodies in different histological compartments in a biopsy from a normal human intestine (Figure 3). The field of view (360 × 360 μm2) has been obtained by merging 16 different panels collected with a sampling pixel of about 80 nm. Such an analysis suggested a correlation between intracellular properties and histological compartmentalisation. In vitro studies attributed to PML an anti-proliferative effect. Mean size and intensity of PML nuclear bodies in the intestine crypts is lower than the values calculated in the surrounding connective tissue, suggesting a correlation between protein content and proliferation, the crypts being sites of high replicative activity.
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